Methods for preparing a purified maytansinoid conjugate in a solution

ABSTRACT

This invention describes a method of conjugating a cell binding agent such as an antibody with an effector group (e.g., a cytotoxic agent) or a reporter group (e.g., a radionuclide), whereby the reporter or effector group is first reacted with a bifunctional linker and the mixture is then used without purification for the conjugation reaction with the cell binding agent. The method described in this invention is advantageous for preparation of stably-linked conjugates of cell binding agents, such as antibodies with effector or reporter groups. This conjugation method provides in high yields conjugates of high purity and homogeneity that are without inter-chain cross-linking and inactivated linker residues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No.16/265,249, filed Feb. 1, 2017, which is a continuation of Ser. No.15/694,164, filed Sep. 1, 2017, now U.S. Pat. No. 10,233,257, issuedMar. 19, 2019, which is a continuation of U.S. patent application Ser.No. 15/167,476, filed May 27, 2016, now U.S. Pat. No. 9,771,432, issuedSep. 26, 2017, which is a continuation of U.S. patent application Ser.No. 14/095,579, filed Dec. 3, 2013, now U.S. Pat. No. 9,376,500, issuedJun. 28, 2016, which is a continuation of U.S. patent application Ser.No. 12/793,175, filed Jun. 3, 2010, now U.S. Pat. No. 8,624,003, issuedJan. 7, 2014, which claims the benefit of U.S. Provisional PatentApplication No. 61/183,774, filed Jun. 3, 2009, the entire contents ofeach which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a novel method of conjugating an effectorgroup (e.g., a cytotoxic agent) or a reporter group (e.g., a radiolabel)to a cell binding agent, such as an antibody or a fragment thereof, viaa bifunctional linker. More specifically, this invention relates to anovel method of conjugating an effector group (e.g., maytansinoids) or areporter group (e.g., a radiolabel) to a cell binding agent (e.g., anantibody or a fragment thereof) via a bifunctional linker such that theprocess eliminates the steps that result in formation of undesiredhydrolyzed species or undesired cross-linked species formed due tointra-molecular or inter-molecular reactions.

BACKGROUND OF THE INVENTION

Conjugates of cell binding agents, such as antibodies, with effectorgroups, such as small cytotoxic agents or cytotoxic proteins, are ofimmense interest for the development of anti-cancer therapeutics(Richart, A. D., and Tolcher, A. W., 2007, Nature Clinical Practice, 4,245-25). These conjugates are tumor-specific due to the high specificityof the selected antibodies toward antigens expressed on the cell surfaceof tumor cells. Upon specific binding to the tumor cell, theantibody-cytotoxic agent conjugate is internalized and degraded insidethe target cancer cell thereby releasing the active cytotoxic agent thatinhibits essential cellular functions such as microtubule dynamics orDNA replication resulting in the killing of the cancer cell. Variouslinkers have been employed to link the antibodies with cytotoxic agentswith the goal of enhancing the delivery of the agent inside the cellupon internalization and processing of the conjugate, while maintainingthe desired stability of the conjugate in plasma. These linkers includedisulfide linkers designed with different degrees of steric hindrance toinfluence their reduction kinetics with intracellular thiol, cleavablepeptide linkers such as valine-citrulline linkage, and non-cleavablelinkers such as thioether linkage (Widdison, W., et al., J. Med. Chem.,2006, 49, 4392-4408; Erickson, H., et al., Cancer Res., 2006, 66,4426-4433).

Conjugates of cell binding agents such as antibodies with labels orreporter groups are useful for tumor-imaging applications in cancerpatients, immunoassay applications for diagnosis of various diseases,cancer therapy using radioactive nuclide-ligand conjugates, and affinitychromatography applications for purification of bioactive agents such asproteins, peptides, and oligonucleides. The labels or reporter groupsthat are conjugated with cell-binding agents include fluorophores, andaffinity labels such as biotin.

The conventional method of conjugation of the cell-binding agent such asan antibody (Ab) with an effector group (e.g., a cytotoxic agent) or areporter group (e.g., a radiolabel) linked via a non-reducible linkage(such as thioether linkage) employs two distinct reaction steps with theantibody and necessitates the use of purification steps. In the firstreaction step, the antibody is reacted with a heterobifunctional linkerbearing two different reactive groups (e.g., X and Y). For example, inone approach, the reaction of an antibody's reactive residues (such aslysine amino residues) with the X reactive group (such asN-hydroxysuccinimide ester) of the heterobifunctional reagent results inthe incorporation of the linker with Y reactive group at one or morereactive residues in the antibody (such as lysine amino residues). Theinitially modified antibody product must be purified from the excesslinker or hydrolyzed linker reagent before the next step can occur. Inthe second reaction step, the linker-modified antibody containing the Yreactive group (such as maleimide or haloacetamide) is reacted with theeffector such as an effector group (C) (e.g., a cytotoxic agent)containing a reactive group such as thiol to generate theantibody-effector conjugate, which is again purified in an additionalpurification step (see, e.g., U.S. Pat. Nos. 5,208,020, 5,416,064, or5,024,834). Thus, in the above process, at least two purification stepsare needed.

Another approach that involves two reaction and purification steps toconjugate antibody with an effector or reporter group uses the reactionof thiol residues in antibody (generated via modification of antibodywith thiol-generating reagents such as 2-iminothiolane, or viamutagenesis to incorporate non-native cysteine residues, or viareduction of native disulfide bonds) with a homobifunctional linkerY-L-Y containing Y reactive groups (such as maleimide or haloacetamide).

Major drawbacks of incorporating a reactive group Y such as maleimide(or haloacetamide) in an antibody or peptide are the propensity of thereactive maleimide (or haloacetamide) groups to undergo intra- orinter-molecular reaction with the native histidine, lysine, tyrosine, orcysteine residues in antibody or peptide (Papini, A. et al., Int. J.Pept. Protein Res., 1992, 39, 348-355; Ueda, T. et al., Biochemistry,1985, 24, 6316-6322), and aqueous inactivation of the Y maleimide group.The undesired intra-molecular or inter-molecular reaction of maleimide(or haloacetamide) groups Y incorporated in antibody with the nativehistidine, lysine, or cysteine residues in antibody, and aqueousinactivation of the Y maleimide group before the second reaction withthe effector or reporter group C give rise to cross-linked proteins orheterogeneous conjugates and lower the efficiency of the second reactionwith the effector or reporter group C. The heterogeneous conjugateproduct—cross-linked protein or peptide generated from the undesiredreaction of the initially incorporated group Y (such as maleimide group)with native groups in the antibody or peptides (such as histidine,lysine, tyrosine, or cysteine), or with inactive maleimide residuesgenerated by aqueous inactivation—may have inferior activity andstability than the desired homogeneous conjugate product.

Processes for conjugating antibodies to thiol-containing cytotoxicagents via disulfide linkages have been described previously (see, e.g.,U.S. Pat. Nos. 5,208,020, 5,416,064, 6,441,163, U.S. Patent PublicationNo. 2007/0048314 A1). These processes involve the initial reaction ofantibody with a heterobifunctional reagent, followed by a secondreaction with a thiol-containing cytotoxic agent. An alternative processhas been described in U.S. Pat. No. 6,441,163 B1 in which thedisulfide-linked reactive ester of the cytotoxic agent is first purifiedand then reacted with the antibody, but which involves an additionalreaction and purification step starting from the thiol group-containingcytotoxic agent before the reaction step with the antibody.

A further drawback of the current process to make conjugates of cellbinding agents is the need for two purification steps, which lowers theoverall yield and also makes the process cumbersome and uneconomical forscale-up.

In view of the foregoing, there is a need in the art to develop improvedmethods of preparing cell-binding agent-drug conjugate compositions thatare of substantially high purity and can be prepared avoiding cumbersomesteps and by reducing time and cost to the user. The invention providessuch a method. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

SUMMARY OF THE INVENTION

The present invention describes a conjugation method for preparingnon-reducible, thioether-linked conjugates of the formula C-L-CBA,wherein C represents an effector or reporter molecule (e.g., a cytotoxicagent or a radiolabel), L is a linker and CBA is a cell binding agent(e.g., an antibody or a fragment thereof), by utilizing a directreaction of the thiol-containing cytotoxic agent (e.g., maytansinoids)with a hetero-or a homo-bifunctional reagent, (e.g., cleavable or anon-cleavable linker) followed by mixing of the unpurified reactionmixture with a cell binding agent (e.g., an antibody or a fragmentthereof), thereby generating the non-reducible, thioether-linkedconjugate by a process that is more efficient, has a high yield, and isamenable for scale up. Another important advantage is that suchconjugation method yields thioether-linked non-reducible conjugates withno inter-chain protein cross-linking or inactivated residues (e.g.,maleimide or haloacetamide residues). The novel methods disclosed inthis application can be applied to the preparation of any conjugaterepresented by the above formula.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows conjugation of antibody with a reaction mixture of themaytansinoid DM1 (or DM4) and Maleimide-PEG_(n)-NHS linker

FIG. 2 shows reducing SDS-PAGE of Ab-(PEG₄-Mal)-DM4 conjugates preparedusing the method described in this invention versus conjugates preparedusing the traditional 2-step method. Each sample lane contained 10 μgprotein; the gel was stained with Coomassie Blue. Lanes 1 and 2contained molecular weight markers. Lane 3 contained conjugate preparedby the traditional two-step method with 6.1 DM4 per Ab. Lane 4 containedconjugate prepared by the method described in this invention andcontained 6.2 DM4 per Ab.

FIGS. 3A-B show Protein LabChip electrophoresis of Ab-(PEG₄-Mal)-DM4conjugates prepared using the method described in this invention versusconjugates prepared using the traditional 2-step method. FIG. 3A showsProtein LabChip electrophoresis under reducing condition (Agilent 2100Bioanalyzer/Agilent Protein 230 kit) of Ab-(PEG₄-Mal)-DM4 conjugates.Lane 1: molecular weight markers; lane 2: Ab-PEG₄-Mal-DM4, 6.2 D/Ab,synthesized using the method described in this invention; lane 3:Ab-PEG₄-Mal-DM4, 6.1 D/Ab, synthesized using the 2 step conjugationmethod; lane 4: unconjugated Ab (0.24 microgram total protein in eachlane). The upper marker, system peak and lower marker bands representexternal markers added from kit. FIG. 3B shows the quantitation ofprotein bands from Protein LabChip electrophoresis.

FIGS. 4A-B show MS of Ab-(PEG₄-Mal)-DM4 conjugates prepared using themethod described in this invention versus conjugates prepared using thetraditional 2-step method. FIG. 4A shows MS of conjugate prepared by thetraditional two-step method with 6.1 DM4 per Ab. Due to significantheterogeneity of the conjugate the MS peaks could not be resolved well.FIG. 4B shows MS of conjugate prepared by the method described in thisinvention and contained 6.2 DM4 per Ab. Due to homogeneity of theconjugate, the MS peaks were well resolved.

FIG. 5 shows binding of an anti-CanAg antibody-PEG₄-Mal-DM1 conjugatewith 6.7 DM1 per antibody (prepared using the method described in thisinvention) versus binding of unmodified antibody toward CanAgantigen-expressing COLO205 cells. The binding was measured influoresence units.

FIG. 6 shows in vitro cytotoxicity of an anti-CanAgAntibody-PEG₄-Mal-DM1 conjugate with 6.7 DM1 per antibody (preparedusing the method described in this invention) toward CanAgantigen-expressing COLO205 cells. The conjugate was added to COLO205cells, and after 5 days of continuous incubation with the conjugate, theviability of the cells was measured using WST-8 assay. A controlexperiment to demonstrate the specificity of the conjugate was carriedout using an excess of unconjugated anti-CanAg antibody to block thebinding and cytotoxicity of the conjugate toward target cancer cells.

FIG. 7 shows conjugation of antibody with a reaction mixture of DM1 (orDM4) and Maleimide-Sulfo-NHS linker.

FIG. 8 shows reducing SDS-PAGE of Ab-(Sulfo-Mal)-DM1 conjugates preparedusing the method described in this invention versus conjugates preparedusing the traditional 2-step method. Each sample lane contained 10 μgprotein; the gel was stained with Coomassie Blue. Lane 1 containedmolecular weight markers. Lanes 3 and 5 contained conjugates prepared bythe method described in this invention and contained 3.6 and 5.6 DM1 perAb, respectively. Lanes 2 and 4 contained conjugates prepared by thetraditional two-step method and contained 4.0 and 5.7 DM1 per Ab,respectively.

FIGS. 9A-B show Protein LabChip electrophoresis of Ab-(Sulfo-Mal)-DM1conjugate prepared using the method described in this invention versusconjugate prepared using the traditional 2-step method. FIG. 9A showsProtein LabChip electrophoresis under reducing condition (Agilent 2100Bioanalyzer/Agilent Protein 230 kit) of Ab-(Sulfo-Mal)-DM1 conjugates.Lane 1: molecular weight markers; lane 2: unconjugated Ab; lane 3:Ab-Sulfo-Mal-DM1, 5.7 D/Ab, synthesized using the 2 step conjugationmethod; lane 4: Ab-Sulfo-Mal-DM1, 5.6 D/Ab, synthesized using the methoddescribed in this invention; 0.22 microgram total protein loaded perwell. The upper marker, system peak and lower marker bands representexternal markers added from kit (0.24 microgram total protein per well).FIG. 9B shows the quantitation of protein bands from Protein LabChipelectrophoresis.

FIGS. 10A-B show LC-MS comparison of Antibody-(Sulfo-Mal)-DM1 conjugateprepared by the method described in this invention versus by thetraditional two-step conjugation method. FIG. 10A shows MS of conjugatewith 3.6 DM1/Ab prepared using the method described in this inventionshows a homogeneous conjugate with 1-6 DM1-bearing discrete conjugatepeaks. FIG. 10B shows MS of conjugate with 4.0 DM1/Ab prepared by thetraditional two-step conjugation method. The MS for the conjugateprepared by the traditional two-step method shows peaks corresponding toconjugates, and conjugates with hydrolyzed or cross-linked linkers (suchas conjugate with 2 DM1, plus one L, 2L, and 3 L), indicating aheterogeneous product.

FIG. 11 shows binding of an anti-CanAg antibody-Sulfo-Mal-DM1 conjugatewith 5.6 DM4 per antibody (prepared using the method described in thisinvention) versus binding of unmodified antibody toward CanAgantigen-expressing COLO205 cells. The binding was measured influoresence units.

FIG. 12 shows in vitro cytotoxicity of an anti-CanAgAntibody-Sulfo-Mal-DM1 conjugate with 5.6 DM4 per antibody (preparedusing the method described in this invention) toward CanAgantigen-expressing COLO205 cells. The conjugate was added to COLO205cells and after 5 days of continuous incubation with the conjugate, theviability of the cells was measured using WST-8 assay. A controlexperiment to demonstrate the specificity of the conjugate was carriedout using an excess of unconjugated anti-CanAg antibody to block thebinding and cytotoxicity of the conjugate toward target cancer cells.

FIG. 13 shows conjugation of antibody with a reaction mixture of DM1 (orDM4) and Sulfo-NHS SMCC linker.

FIG. 14 shows reducing SDS-PAGE of Ab-(SMCC)-DM1 conjugate preparedusing the method described in this invention versus conjugate preparedusing the traditional 2-step method. Each sample lane contained 10microgram total protein; the gel was stained with Coomassie Blue. Lane 1contains molecular weight markers, Lane 2 contains unconjugated Ab, Lane3 contains conjugate prepared by the traditional two-step method with3.1 DM1 per Ab and Lane 4 contains conjugate prepared by the methoddescribed in this invention with 3.1 DM1 per Ab.

FIGS. 15A-B show Protein LabChip electrophoresis of Ab-(SMCC)-DM1conjugate prepared using the method described in this invention versusconjugate prepared using the traditional 2-step method. FIG. 15A showsProtein LabChip electrophoresis under reducing condition (Agilent 2100Bioanalyzer/Agilent Protein 230 kit) of Ab-SMCC-DM1 conjugates. Lane 1:molecular weight markers; lane 2: Ab-SMCC-DM1, 3.1 D/Ab, synthesizedusing the method described in this patent; lane 3: unconjugated Ab; lane4: Ab-SMCC-DM1, 3.1 D/Ab, synthesized using the 2 step conjugationmethod; (0.24 microgram total protein in each lane). The upper marker,system peak and lower marker bands represent external markers added fromkit. FIG. 15B shows the quantitation of protein bands from ProteinLabChip electrophoresis.

FIGS. 16A-B show LC-MS comparison of Antibody-(SMCC)-DM1 conjugateprepared by the method described in this invention versus conjugateprepared by the traditional two-step conjugation method. FIG. 16A showsMS of conjugate prepared by the sequential two-step method with 3.1 DM1per Ab. Each major conjugate peak has associated side peaks due to thepresence of hydrolyzed and cross-linked linker fragments. FIG. 16B showsMS of conjugate prepared by the method described in this invention with3.1 DM1 per Ab. Due to homogeneity of the conjugate, the MS peaks werewell resolved.

FIG. 17 shows proposed mechanisms for inter-chain cross-linking andmaleimide inactivation during conjugation by the traditional 2-stepmethod.

FIG. 18 shows non-reducing SDS PAGE of Ab-(Sulfo-Mal)-DM4 conjugateprepared using the method described in this invention and quenching offree DM4 thiol (after the initial DM4+NHS-Sulfo-Mal heterobifunctionalreagent coupling reaction) using 4-maleimidobuytric acid prior to theantibody conjugation reaction. Each sample contained 10 μg protein; thegel was stained with Coomassie Blue. Lanes 1 and 5 contained molecularweight markers. Lane 2 contained Ab alone. Lane 3 contained conjugateprepared by the method described in this invention without addition of4-maleimidobuytric acid. Lane 4 contained conjugate prepared by themethod described in this invention with addition of 4-maleimidobutyricacid after the initial DM4+NHS-Sulfo-Mal heterobifunctional reagent(prior to the antibody conjugation step).

FIG. 19 shows preparation of disulfide-linked conjugate of antibodyusing a reaction mixture of DM1 (or DM4) and SPDB linker.

FIG. 20 shows preparation of antibody-maytansinoid conjugate with bothdisulfide- and non-cleavable PEG₄-Mal linkers via antibody conjugationwith an unpurified reaction mixture of DM1 (or DM4) and both SPDB andNHS-PEG₄-Mal linkers.

FIG. 21 shows MS of antibody-maytansinoid conjugate with both disulfide-and non-cleavable PEG₄-Mal linkers (prepared by conjugation of antibodywith an unpurified reaction mixture of DM1, or DM4, and both SPDB andNHS-PEG₄-Mal linkers).

FIG. 22 shows the conjugation of antibody with a reaction mixture of DM1(or DM4) and SMCC linker.

FIG. 23 shows the MS of antibody-SMCC-DM1 conjugate prepared using SMCCby the method described in this invention, containing average 3.1 DM1per antibody.

FIG. 24 shows the preparation of disulfide-linked conjugate of antibodyusing a reaction mixture of DM1 (or DM4) and SSNPB linker.

FIG. 25 shows the conjugation of antibody with a reaction mixture of DM1(or DM4) and heterobifunctional linker with aliphatic linear carbonchain.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

This invention describes a novel method of conjugating athiol-containing effector (e.g., a cytotoxic agent) or a reporter group(e.g., a radiolabel) with a cell binding agent (e.g., an antibody), inwhich the thiol-group containing effector or reporter is first reactedwith a bifunctional linker reagent in organic, aqueous, or mixedorganic/aqueous solvent, followed by reaction of the unpurified reactionmixture with the cell binding agent in organic, aqueous or mixedorganic/aqueous solvents.

Legend Abbreviations

Abbreviations which have been used in the descriptions of the Schemesand the Examples that follow are:

C=Effector or a reporter group (e.g., a cytotoxic agent or a radiolabel)

L=Linker (e.g., cleavable or a non-cleavable linker)

X=amine-reactive group (e.g., N-hydroxysuccinimide ester (NHS ester),sulfo-NHS ester, p-nitrophenol ester, tetrafluorosulfonate phenyl ester,1-hydroxy-2-nitro-benzene-4-sulfonic acid ester)

Y=Maleimide, or haloacetamide (iodoacetamide, bromoacetamide)

Y_(b) is a reactive mixed disulfide group (e.g., 2-pyridyldithio,4-pyridyldithio, 2-nitro-pyridyldithio, 5-nitro-pyridyldithio,2-carboxy-5-nitro-pyridyldithio)

X′=amide linkage

Y′=thioether (R—S—R′) or selenoether (R—Se—R′) linkage

Y_(b)′=disulfide (R—S—S—R′) linkage

In one embodiment of this invention, a process for the preparation of athioether-linked conjugate of a cell-binding agent with an effector or areporter molecule is described, the process comprising the followingsteps: a) contacting a heterobifunctional linker of formula X-L-Y with athiol-containing effector or reporter molecule C (e.g., a maytansinoidor a radionuclide) in aqueous solvent, organic solvent, or mixedorganic/aqueous reaction mixtures which yields an intermediate productof formula X-L-Y′—C; b) mixing of the reaction mixture withoutpurification with a cell-binding agent such as an antibody (Ab) toproduce a conjugate of formula Ab-(X′-L-Y′—C)_(m), wherein, L is asubstituted or unsubstituted linear, branched or cyclic alkyl, alkenyl,or alkynyl group bearing from 1-10 carbon atoms, a simple or substitutedaryl unit (substituents selected from alkyl, alkoxy, halogen, nitro,fluoro, carboxy, sulfonate, phosphate, amino, carbonyl, piperidino) or apolyethylene glycol containing unit (preferably 1-500 PEG spacer, ormore preferably 1-24 PEG spacer, or still more preferably 2-8 PEGspacer); X and Y are amine or thiol-reactive group such asN-hydroxysuccinimide ester and maleimide or haloacetamide; Ab is anantibody; m is an integer from 1-20; X′ is modified X site (e.g., anamide linkage) upon reaction with antibody; Y′ is modified Y site (e.g.,thioether linkage) upon reaction with, for example, a cytotoxic agent ora radiolabel of the effector or reporter group; and c) purification ofthe conjugate by tangential flow filtration, dialysis, or chromatography(e.g., gel filtration, ion-exchange chromatography, hydrophobicinteraction chromatography) or a combination thereof. Preferably, Y is athiol-reactive group selected from maleimide or haloacetamide.Preferably, L is a linear or branched alkyl group with 1-6 carbons or2-8 PEG spacer. Preferably, C is a cytotoxic agent selected from amaytansinoid, a CC-1065 analog, a taxane, a DNA-binding agent, and morepreferably it is a maytansinoid.

This reaction sequence represented in formulae 1 and 2:X-L-Y+C→X-L-Y′—C   (1)Ab+X-L-Y′—C (unpurified from reaction 1)→Ab-(X′-L-Y′—C)_(m)   (2)does not involve any purification of the intermediate product X-L-Y′—C,and therefore provides the advantage of directly mixing it with theantibody (the unpurified intermediate product is added to the antibodyor, the antibody is added to the unpurified intermediate product)thereby making the method advantageous for conjugation because iteliminates the need of a cumbersome purification step. Importantly, thismethod yields homogeneous conjugate with no inter-chain proteincross-linking or inactivated maleimide residues, in contrast to theinter-chain protein cross-linking and inactivated maleimide residuesobserved in conjugates prepared by the traditional two step reaction andpurification sequence.

The reaction 1 can be carried out at high concentrations of theheterobifunctional linker, X-L-Y, and the effector or reporter group Cin aqueous solvent, organic solvent, or organic/aqueous reactionmixtures, resulting in faster reaction rates than at lowerconcentrations in aqueous solutions for conjugates prepared by thetraditional two step reaction and purification sequence.

The intermediate product X-L-Y′—C generated in reaction 1 can be storedunpurified in a frozen state, at low temperatures in aqueous solvent atappropriate low pH (e.g., pH ˜4-6), in organic solvents, or in mixedorganic/aqueous mixtures, or in lyophilized state, for prolonged periodsand can be mixed later with the antibody solution for the finalconjugation reaction at a higher pH value of about 4-9, therefore addingto the convenience of this reaction sequence. The intermediate productcan be diluted as required with organic solvent or with aqueous buffer,or a mixture of organic solvent and aqueous buffer prior to mixing withthe cell binding agent. The term “about” as used herein in connectionwith a numerical should be understood to refer to all such numbers,including all numbers and small variations therefrom. The reaction ofthe intermediate product X-L-Y′—C with antibody can be carried out at pHvalues of about 4 to about pH 9, preferably in the pH range of about 5to 8.7, more preferably, in the pH range of about 6.5 to about 8.5, suchas, pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, and 8.5, a pH range therein or smallvariations therefrom. The buffers used for the reaction of the antibodywith the intermediate product X-L-Y′—C in the preferential pH range ofabout 6.5 to 8.5 are buffers with pK_(a) values around this pH range,such as phosphate and HEPES buffer. These preferred buffers should nothave primary or secondary amino groups, or other reactive groups, thatcan react with linker X (such as N-hydroxysuccinimide ester).

A stoichiometric or a slight excess of C over the heterobifunctionallinker X-L-Y is used in the first reaction to ensure that all Y group(such as maleimide) is reacted before the unpurified mixture is added tothe antibody. An optional additional treatment with a quenching reagent(such as 4-maleimidobutyric acid, 3-maleimidopropionic acid, orN-ethylmaleimide, or iodoacetamide, or iodoacetamidopropionic acid) canbe done to ensure that any unreacted C is quenched before mixing withthe antibody to minimize any unwanted thiol-disulfide interchangereaction with the native antibody disulfide groups. Upon quenching withpolar, charged thiol-quenching reagents (such as 4-maleimidobutyric acidor 3-maleimidopropionic acid), the excess, unreacted C is converted intoa polar, charged adduct that can be easily separated from thecovalently-linked conjugate. Optionally, the final reaction mixture 2,before purification, is treated with nucleophiles, such as amino groupcontaining nucleophiles (e.g., lysine, taurine, hydroxylamine) to quenchany unreacted linker (X-L-Y′—C).

An alternative method for the reaction of antibody with the unpurifiedinitial reaction mixture of maytansinoids (DMx) and heterobifunctionallinker involves mixing the initial reaction mixture of DMx andheterobifunctional linker (upon completion of the DMx-linker reaction)with antibody at low pH (pH ˜5) followed by addition of buffer or baseto increase the pH to about 6.5-8.5 for the conjugation reaction.

This new method is applied to the preparation of an antibody conjugatewith the cytotoxic maytansinoid drug. The antibody-maytansinoidconjugates prepared using this method outlined in the reaction sequence1-2 unexpectedly were much superior in homogeneity compared to theconjugates prepared by the traditional two step reaction andpurification sequence, based on characterization of the conjugates byreducing SDS-PAGE, protein LabChip electrophoresis, and massspectrometry. The conjugation method described in this invention thatinvolves the reaction sequence 1-2 also does not require anyintermediate purification step and is therefore significantly moreconvenient than the traditional two-step method.

In a second embodiment of the invention, a process for the preparationof a thioether-linked conjugate of a cell-binding agent with an effectoror reporter molecule is described comprising the following steps: a)contacting a homobifunctional linker of formula Y-L-Y with a thiol- oramine-containing effector or reporter group C (such as a cytotoxicagent) in aqueous solvent, organic solvent, or mixed aqueous/organicreaction mixtures to yield Y-L-Y′—C, b) mixing of the reaction mixturewithout purification with an antibody in aqueous solution oraqueous/organic mixture to produce a conjugate of formulaAb-(Y′-L-Y′—C)_(m), wherein, L is as defined above; Y is a thiol- oramine-reactive group such as a maleimide or haloacetamide, orN-hydroxysuccinimide or sulfo N-hydroxysuccinimide; Ab is an antibody; mis an integer from 1 to 20; Y′ is the modified Y site (such as athioether or amide linkage) upon reaction with antibody or a modified Ysite (such as a thioether or amide linkage) upon reaction with thecytotoxic agent or effector or reporter group, and c) purification ofthe conjugate by tangential flow filtration, dialysis, or chromatography(gel filtration, ion-exchange chromatography, hydrophobic interactionchromatography) or a combination thereof. The reaction sequencerepresented in formulae 3 and 4:Y-L-Y+C Y-L-Y′—C   (3)Ab+Y-L-Y′—C (unpurified from reaction 3)→Ab-(Y′-L-Y′—C)_(m)   (4)does not involve any purification of the intermediate product Y-L-Y′—C,and therefore is an advantageous method for conjugation.

In a third embodiment, a process for the preparation of adisulfide-linked conjugate of a cell binding agent with an effector orreporter molecule is described that comprises of the following steps: a)contacting a heterobifunctional linker of formula X-L-Y_(b) with theeffector or reporter group C (such as a cytotoxic agent) in aqueoussolvent, organic solvent or mixed organic/aqueous reaction mixtures toyield intermediate product X-L-Y_(b)′—C; b) mixing of the reactionmixture without purification with the antibody in an aqueous solution oraqueous/organic mixture to produce a conjugate of formulaAb-(X′-L-Y_(b)′—C)_(m), wherein, L is as described above; Y_(b) is areactive disulfide such as a pyridyl disulfide or a nitro-pyridyldisulfide; X is an amine-reactive group such as N-hydroxysuccinimideester or sulfo N-hydroxysuccinimide ester; Ab is an antibody; m is aninteger from 1 to 20; X′ is modified X site (such as amide linkage) uponreaction with antibody; Y_(b)′ is modified Y_(b) site (disulfide) uponreaction with the cytotoxic agent or effector or reporter group; and c)purification of the conjugate by tangential flow filtration, dialysis,or chromatography (gel filtration, in-exchange chromatography,hydrophobic interaction chromatography) or a combination thereof. Thereaction sequence is represented in formulae 5 and 6:X-L-Y_(b)+C X-L-Y_(b)′—C   (5)Ab+X-L-Y_(b)′—C (unpurified from reaction 5)→Ab-(X′-L-Y_(b)′—C)_(m)  (6)

In a fourth embodiment, a process for the preparation of conjugates ofantibody with effector or reporter groups with two types oflinkers—non-cleavable (thioether linkage) and cleavable (disulfidelinkage)—comprising the following steps is described: a) contactingX-L-Y and X-L-Y_(b) linkers with the cytotoxic agent C to generateintermediate compounds of formulae X-L-Y′—C and X-L-Y_(b)′—C, b) mixingof the reaction mixtures without purification with the antibody eitherin a sequence or simultaneously as indicated in reaction formulae 7-9:X-L-Y+C X-L-Y′—C   (7)X-L-Y_(b)+C X-L-Y_(b)′—C   (8)Ab+X-L-Y′—C+X-L-Y_(b)′—C (unpurified from reactions7-8)→Ab-(X′-L-Y′—C)_(m)(X′-L-Y_(b)′—C)_(m′)  (9)to provide a conjugate Ab-(X′-L-Y′—C)_(m)(X′-L-Y_(b)′—C)_(m), wherein,the definitions of X, L, Y′, C, Y_(b)′, and m are as given above, and m′is an integer from 1 to 20; and c) purification of the conjugate bytangential flow filtration, dialysis, or chromatography (gel filtration,ion-exchange chromatography, hydrophobic interaction chromatography) ora combination thereof. These two linker effector intermediates (X-L-Y′—Cand X-L-Y_(b)′—C) are mixed without purification with the antibody indifferent sequences (first X-L-Y′—C then X-L-Y_(b)′—C, or firstX-L-Y_(b)′—C then X-L-Y′—C or simultaneously) in various ratio.

The reactions 1, 3, 5, and 7-8, can be carried out at highconcentrations of the bifunctional linker (X-L-Y, X-L-Y_(b), or Y-L-Y)and the effector or reporter group C in aqueous solvent, organicsolvent, or organic/aqueous reaction mixtures, resulting in fasterreaction rates than at lower concentrations in aqueous solutions forconjugates prepared by the traditional two step reaction andpurification sequence where the solubility of reagents is limiting.

The intermediate products X-L-Y′—C, or Y-L-Y′—C, or X-L-Y_(b)′—Cgenerated in reactions 1, 3, 5, and 7-8 can be stored unpurified in afrozen state, at low temperatures in aqueous solvent at appropriate pH,in organic solvents, or in mixed organic/aqueous mixtures, or inlyophilized state, for prolonged periods and can be added later to theantibody solution for the final conjugation reaction, therefore addingto the convenience of this reaction sequence.

A stoichiometric or a slight excess of C over the heterobifunctionallinker X-L-Y, or Y-L-Y, or X-L-Y_(b) is used in the first reaction toensure that all Y group (such as maleimide) is reacted before theunpurified mixture is added to the antibody. An optional additionaltreatment with a quenching reagent (such as 4-maleimidobutyric acid, or3-maleimidopropionic acid, or N-ethylmaleimide, or iodoacetamide, oriodoacetic acid) can be done to ensure that any unreacted group (such asthiol) in C is quenched before the addition to the antibody to minimizeany unwanted thiol-disulfide interchange reaction with the nativeantibody disulfide groups. The quenching of the excess C using acharged, polar thiol-quenching reagent, after the initial reaction of Cwith the bifunctional linker, converts excess C into a highly polar,water-soluble adduct that is easily separated from the covalently-linkedconjugate by gel filtration, dialysis, or TFF. The final conjugateproduct does not contain any non-covalently associated C. Optionally,the final reaction mixtures 2, 4, 6, and 9, before purification, aretreated with nucleophiles, such as, amino group containing nucleophiles(e.g., lysine, taurine, hydroxylamine) to quench any unreacted linkers(X-L-Y′—C, Y-L-Y′—C, or X-L-Y_(b)′—C).

An alternate method of the reaction of antibody with the unpurifiedinitial reaction mixture of DMx and bifunctional linker involves mixingthe initial reaction mixture of DMx and bifunctional linker (uponcompletion of the DMx-linker reaction) with antibody at low pH (pH ˜5)followed by addition of buffer or base to increase the pH to about6.5-8.5 for the conjugation reaction.

Multiple copies of more than one type of effector can be conjugated tothe antibody by adding two or more linker-effector intermediates derivedfrom two or more different effectors, without purification, to theantibody either in a sequence or simultaneously.

Effector Group(s)

The terms Effector group or Effector molecule are used interchangeablyand the term “Effector group(s)” or “Effector molecule(s)”, as usedherein, is meant to include cytotoxic agents. In certain respects, itmay be desirable that the effector groups or molecules are attached byspacer arms of various lengths to reduce potential steric hindrance.Multiple copies of more than one type of effector can be conjugated tothe antibody by adding two or more linker-effector intermediates derivedfrom two or more different effectors, without purification, to theantibody either in a sequence or simultaneously.

Cytotoxic agents that can be used in the present invention includechemotherapeutic agents or structural analogues of chemotherapeuticagents. “Chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines, such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics, such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin .gamma1 and calicheamicin thetaI, see, e.g., Angew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites, such as methotrexate and5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimnidineanalogs such as, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens, such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals, such asaminoglutethimide, mitotane, trilostane; folic acid replenisher, such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids, such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;siRNA and pharmaceutically acceptable salts, acids or derivatives of anyof the above. Other chemotherapeutic agents that can be used with thepresent invention are disclosed in US Publication No. 20080171040 or USPublication No. 20080305044 and are incorporated in their entirety byreference.

In a preferred embodiment, chemotherapeutic cytotoxic agents areessentially small molecule cytotoxic agents. A “small molecule drug” isbroadly used herein to refer to an organic, inorganic, or organometalliccompound that may have a molecular weight of for example 100 to 1500,more suitably from 120 to 1200, favorably from 200 to 1000, andtypically having a molecular weight of less than about 1000. Smallmolecule cytotoxic agents of the invention encompass oligopeptides andother biomolecules having a molecular weight of less than about 1000.Small molecule cytotoxic agents are well characterized in the art, suchas in WO05058367A2, European Patent Application Nos. 85901495 and8590319, and in U.S. Pat. No. 4,956,303, among others and areincorporated in their entirety by reference.

Preferable small molecule cytotoxic agents are those that allow forlinkage to the cell-binding agent. The invention includes knowncytotoxic agents as well as those that may become known. Especiallypreferred small molecule cytotoxic agents include cytotoxic agents.

The cytotoxic agent may be any compound that results in the death of acell, or induces cell death, or in some manner decreases cell viability,wherein each cytotoxic agent comprises a thiol moiety.

Preferred cytotoxic agents are maytansinoid compounds, taxane compounds,CC-1065 compounds, daunorubicin compounds and doxorubicin compounds,pyrrolobenzodiazepine dimers, calicheamicins, auristatins and analoguesand derivatives thereof, some of which are described below.

Other cytotoxic agents, which are not necessarily small molecules, suchas siRNA, are also encompassed within the scope of the instantinvention. For example, siRNAs can be linked to the crosslinkers of thepresent invention by methods commonly used for the modification ofoligonucleotides (see, for example, US Patent Publications 20050107325and 20070213292). Thus the siRNA in its 3′ or 5′-phosphoromidite form isreacted with one end of the crosslinker bearing a hydroxyl functionalityto give an ester bond between the siRNA and the crosslinker. Similarlyreaction of the siRNA phosphoramidite with a crosslinker bearing aterminal amino group results in linkage of the crosslinker to the siRNAthrough an amine. siRNA are described in detail in U.S. PatentPublication Numbers: 20070275465, 20070213292, 20070185050, 20070161595,20070054279, 20060287260, 20060035254, 20060008822, 20050288244,20050176667, which are incorporated herein in their entirety byreference.

Maytansinoids

Maytansinoids that can be used in the present invention are well knownin the art and can be isolated from natural sources according to knownmethods or prepared synthetically according to known methods.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogues. Examples of suitable maytansinol analogues include thosehaving a modified aromatic ring and those having modifications at otherpositions.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamitocin P2);

(2) C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H2S or P2S5);

(2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudiflora);

(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

The synthesis of thiol-containing maytansinoids useful in the presentinvention is fully disclosed in U.S. Pat. Nos. 5,208,020, 5,416,064, andU.S. Patent Application No. 20040235840.

Maytansinoids with a thiol moiety at the C-3 position, the C-14position, the C-15 position or the C-20 position are all expected to beuseful. The C-3 position is preferred and the C-3 position ofmaytansinol is especially preferred. Also preferred are anN-methyl-alanine-containing C-3 thiol moiety maytansinoid, and anN-methyl-cysteine-containing C-3 thiol moiety maytansinoid, andanalogues of each.

Specific examples of N-methyl-alanine-containing C-3 thiol moietymaytansinoid derivatives useful in the present invention are representedby the formulae M1, M2, M3, M6 and M7.

wherein:

-   l is an integer of from 1 to 10; and-   May is a maytansinoid.

wherein:

-   R₁ and R₂ are H, CH₃ or CH₂CH₃, and may be the same or different;-   m is 0, 1, 2 or 3; and-   May is a maytansinoid.

wherein:

-   n is an integer of from 3 to 8; and-   May is a maytansinoid.

wherein:

-   l is 1, 2 or 3;-   Y₀ is Cl or H; and-   X₃ is H or CH₃.

wherein:

-   R₁, R₂, R₃, R₄ are H, CH₃ or CH₂CH₃, and may be the same or    different;-   m is 0, 1, 2 or 3; and-   May is a maytansinoid.

Specific examples of N-methyl-cysteine-containing C-3 thiol moietymaytansinoid derivatives useful in the present invention are representedby the formulae M4 and M5.

wherein:

-   o is 1, 2 or 3;-   p is an integer of 0 to 10; and-   May is a maytansinoid.

wherein:

-   o is 1, 2 or 3;-   q is an integer of from 0 to 10;-   Y₀ is Cl or H; and-   X₃ is H or CH₃.

Preferred maytansinoids are those described in U.S. Pat. Nos. 5,208,020;5,416,064; 6,333.410; 6,441,163; 6,716,821; RE39,151 and 7,276,497.

Taxanes

The cytotoxic agent according to the present invention may also be ataxane.

Taxanes that can be used in the present invention have been modified tocontain a thiol moiety. Some taxanes useful in the present inventionhave the formula T1 shown below:

Preferred taxoids are those described in U.S. Pat. Nos. 6,340,701;6,372,738; 6,436,931; 6,596,757; 6,706,708; 7,008,942; 7,217,819 and7,276,499.

CC-1065 Analogues

The cytotoxic agent according to the present invention may also be aCC-1065 analogue.

According to the present invention, the CC-1065 analogues contain an Asubunit and a B or a B-C subunit. Preferred CC-1065 analogs are thosedescribed in U.S. Pat. Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660;6,586,618; 6,756,397 and 7,049,316.

Daunorubicin/Doxorubicin Analogues

The cytotoxic agent according to the present invention may also be adaunorubicin analogue or a doxorubicin analogue.

The daunorubicin and doxorubicin analogues of the present invention canbe modified to comprise a thiol moiety. The modifieddoxorubicin/daunorubicin analogues of the present invention, which havea thiol moiety, are described in WO 01/38318. The modifieddoxorubicin/daunorubicin analogues can be synthesized according to knownmethods (see, e.g., U.S. Pat. No. 5,146,064).

Auristatin include auristatin E, auristatin EB (AEB), auristatin EFP(AEFP), monomethyl auristatin E (MMAE) and are described in U.S. Pat.No. 5,635,483, Int. J. Oncol. 15:367-72 (1999); Molecular CancerTherapeutics, vol. 3, No. 8, pp. 921-932 (2004); U.S. application Ser.No. 11/134826. U.S. Patent Publication Nos. 20060074008, 2006022925.

The cytotoxic agents according to the present invention includepyrrolobenzodiazepine dimers that are known in the art (U.S. Pat. Nos7,049,311; 7,067,511; 6,951,853; 7,189,710; 6,884,799; 6,660,856).

Analogues and Derivatives

One skilled in the art of cytotoxic agents will readily understand thateach of the cytotoxic agents described herein can be modified in such amanner that the resulting compound still retains the specificity and/oractivity of the starting compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thecytotoxic agents described herein. Thus, the cytotoxic agents of thepresent invention include analogues and derivatives of the compoundsdescribed herein.

Reporter Group(s)

The terms Reporter group or Reporter molecule are used interchangeablyand the term “Reporter group(s)” or “Reporter molecule(s)”, as usedherein, refers to a substance which is delivered to the specificsubstance or cells by the specific affinity portion of the reagent, fora diagnostic or therapeutic purpose; examples are radioisotopes,paramagnetic contrast agents, and anti-cancer agents. Various labels orreporter groups are useful for tumor-imaging applications in cancerpatients, immunoassay applications for diagnosis of various diseases,cancer therapy using radioactive nuclide-ligand conjugates, and affinitychromatography applications for purification of bioactive agents such asproteins, peptides, and oligonucleides. The labels or reporter groupsthat are conjugated with cell-binding agents include fluorophores, andaffinity labels such as biotin. Such reporter group references can befound in US publication number 2007/0092940. Reporter groups including,for example, biotin or fluorescein can also be attached to a PEGconjugate moiety. A number of suitable reporter groups are known in theart, e.g., U.S. Pat. No. 4,152,411 and Hirschfeld U.S. Pat. Nos.4,166,105, 5,223,242, 5,501,952, US publication 20090136940 and areincorporated in their entirety by reference.

Linkers

The conjugates may be prepared by in vitro methods. In order to link adrug to the cell-binding agent, a linking group is used. Suitablelinking groups are well known in the art and include non-cleavable orcleavable linkers. A non-cleavable linker is any chemical moiety that iscapable of linking a cytotoxic agent to a cell-binding agent in astable, covalent manner. Non-cleavable linkers are substantiallyresistant to acid-induced cleavage, light-induced cleavage,peptidase-induced cleavage, esterase-induced cleavage, and disulfidebond cleavage. Examples of non-cleavable linkers include linkers havingan N-succinimidyl ester, N-sulfosuccinimidyl ester moiety, maleimido- orhaloacetyl-based moiety for reaction with the drug, the reporter groupor the cell binding agent. Crosslinking reagents comprising amaleimido-based moiety include N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), κ-maleimidoundecanoicacid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester (AMAS),succinimidyl-6-((3-maleimidopropionamido)hexanoate (SMPH),N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI). Cross-linking reagentscomprising a haloacetyl-based moiety includeN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl3-(bromoacetamido)propionate (SBAP).

Other crosslinking reagents lacking a sulfur atom that formnon-cleavable linkers can also be used in the inventive method. Suchlinkers can be derived from dicarboxylic acid based moieties. Suitabledicarboxylic acid based moieties include, but are not limited to,α,ω-dicarboxylic acids of the general formula shown below:HOOC—X₁—Y_(n)—Z_(m)—COOH

wherein X is a linear or branched alkyl, alkenyl, or alkynyl grouphaving 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl groupbearing 3 to 10 carbon atoms, Z is a substituted or unsubstitutedaromatic group bearing 6 to 10 carbon atoms, or a substituted orunsubstituted heterocyclic group wherein the hetero atom is selectedfrom N, O or S, and wherein l, m, and n are each 0 or 1, provided thatl, m, and n are all not zero at the same time.

Many of the non-cleavable linkers disclosed herein are described indetail in U.S. Patent publication number 20050169933.

Cleavable linkers are linkers that can be cleaved under mild conditions,i.e. conditions under which the activity of the cytotoxic agent is notaffected. Many known linkers fall in this category and are describedbelow.

Acid-labile linkers are linkers cleavable at acid pH. For example,certain intracellular compartments, such as endosomes and lysosomes,have an acidic pH (pH 4-5), and provide conditions suitable to cleaveacid-labile linkers.

Linkers that are photo-labile are useful at the body surface and in manybody cavities that are accessible to light. Furthermore, infrared lightcan penetrate tissue.

Some linkers can be cleaved by peptidases. Only certain peptides arereadily cleaved inside or outside cells, see e.g. Trouet et al., 79Proc. Natl. Acad. Sci. USA, 626-629 (1982), Umemoto et al. 43 Int. J.Cancer, 677-684 (1989), and lysosomal-hydrolase cleavablevaline-citrulline linkage (U.S. Pat. No. 6,214,345 B1). Furthermore,peptides are composed of .alpha.-amino acids and peptidic bonds, whichchemically are amide bonds between the carboxylate of one amino acid andthe .alpha.-amino group of a second amino acid. Other amide bonds, suchas the bond between a carboxylate and the .epsilon.-amino group oflysine, are understood not to be peptidic bonds and are considerednon-cleavable.

Some linkers can be cleaved by esterases. Again only certain esters canbe cleaved by esterases present inside or outside cells. Esters areformed by the condensation of a carboxylic acid and an alcohol. Simpleesters are esters produced with simple alcohols, such as aliphaticalcohols, and small cyclic and small aromatic alcohols. For example, thepresent inventors found no esterase that cleaved the ester at C-3 ofmaytansine, since the alcohol component of the ester, maytansinol, isvery large and complex.

Preferred cleavable linker molecules include, for example,N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see, e.g., Carlssonet al., Biochem. J., 173: 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see,e.g., CAS Registry number 341498-08-6), and other reactivecross-linkers, such as those described in U.S. Pat. No. 6,913,748, whichis incorporated herein in its entirety by reference.

Other linkers which can be used in the present invention include chargedlinkers or hydrophilic linkers and are described in U.S. patentapplication Ser. Nos. 12/433,604 and 12/433,668, respectively, which areincorporated herein in its entirety by reference.

Cell Binding Agents

The cell-binding agents used in this invention are proteins (e.g.,immunoglobulin and non-immunoglobulin proteins) which bind specificallyto target antigens on cancer cells. These cell-binding agents include:

antibodies including:

resurfaced antibodies (U.S. Pat. No. 5,639,641);

humanized or fully human antibodies (Humanized or fully human antibodiesare selected from, but not limited to, huMy9-6, huB4, huC242, huN901,DS6, CD38, IGF-IR, CNTO 95, B-B4, trastuzumab, bivatuzumab,sibrotuzumab, and rituximab (see, e.g., U.S. Pat. Nos. 5,639,641,5,665,357, and 7,342,110, International Patent Application WO 02/16,401,U.S. publication number 20060045877, U.S. publication number20060127407, U.S. publication number 20050118183, Pedersen et al.,(1994) J. Mol. Biol. 235, 959-973, Roguska et al., (1994) Proceedings ofthe National Academy of Sciences, Vol 91, 969-973, Colomer et al.,Cancer Invest., 19: 49-56 (2001), Heider et al., Eur. J. Cancer, 31A:2385-2391 (1995), Welt et al., J. Clin. Oncol., 12: 1193-1203 (1994),and Maloney et al., Blood, 90: 2188-2195 (1997)); and

fragments of antibodies such as sFv, Fab, Fab′, and F(ab′)2 thatpreferentially bind to a target cell (Parham, J. Immunol. 131:2895-2902(1983); Spring et al, J. Immunol. 113:470-478 (1974); Nisonoff et al,Arch. Biochem. Biophys. 89:230-244 (1960));

Additional cell-binding agents include other cell binding proteins andpolypeptides exemplified by, but not limited to:

Ankyrin repeat proteins (DARPins; Zahnd et al., J. Biol. Chem., 281, 46,35167-35175, (2006); Binz, H. K., Amstutz, P. & Pluckthun, A. (2005)Nature Biotechnology, 23, 1257-1268) or ankyrin-like repeats proteins orsynthetic peptides described, for example, in U.S. publication number20070238667; U.S. Pat. No. 7,101,675; WO/2007/147213; WO/2007/062466);

interferons (e.g. α, β, γ);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyrotropin releasing hormones), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens; and

growth factors and colony-stimulating factors such as EGF, TGF-α, IGF-1,G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158 (1984)).

Where the cell binding agent is an antibody it binds to an antigen thatis a polypeptide and may be a transmembrane molecule (e.g. receptor) ora ligand such as a growth factor. Exemplary antigens include moleculessuch as renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor vmc, factor IX, tissue factor (TF), and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-α and -β;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha); aserum albumin such as human serum albumin; Muellerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; a microbial protein, such asbeta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen(CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growthfactor (VEGF); receptors for hormones or growth factors; protein A or D;rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-beta1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD40;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe HIV envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM, alpha-V subunit of a heterodimeric human integrinreceptor; a tumor associated antigen such as HER2, HER3 or HER4receptor; and fragments of any of the above-listed polypeptides.

Preferred antigens for antibodies encompassed by the present inventioninclude CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD46;members of the ErbB receptor family such as the EGF receptor, HER2, HER3or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150.95,VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin, and alpha v/beta3 integrinincluding either alpha or beta subunits thereof (e.g. anti-CD11a,anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; tissuefactor (TF); TGF-β; alpha interferon (alpha-IFN); an interleukin, suchas IL-8; IgE; blood group antigens Apo2, death receptor; flk2/flt3receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C etc.The most preferred targets herein are IGF-IR, CanAg, EGF-R, EphA2, MUC1,MUC16, VEGF, TF, CD19, CD20, CD22, CD33, CD37, CD38, CD40, CD44, CD56,CD138, CA6, Her2/neu, CRIPTO (a protein produced at elevated levels in amajority of human breast cancer cells), alpha v/beta3 integrin, alphav/beta5 integrin, TGF-β, CD11a, CD18, Apo2 and C24.

Monoclonal antibody techniques allow for the production of specificcell-binding agents in the form of monoclonal antibodies. Particularlywell known in the art are techniques for creating monoclonal antibodiesproduced by immunizing mice, rats, hamsters or any other mammal with theantigen of interest such as the intact target cell, antigens isolatedfrom the target cell, whole virus, attenuated whole virus, and viralproteins such as viral coat proteins. Sensitized human cells can also beused. Another method of creating monoclonal antibodies is the use ofphage libraries of sFv (single chain variable region), specificallyhuman sFv (see, e.g., Griffiths et al, U.S. Pat. No. 5,885,793;McCafferty et al, WO 92/01047; Liming et al, WO 99/06587.)

Selection of the appropriate cell-binding agent is a matter of choicethat depends upon the particular cell population that is to be targeted,but in general monoclonal antibodies and fragments thereof thatpreferentially bind to a target cell are preferred, if an appropriateone is available.

For example, the monoclonal antibody My9 is a murine IgG_(2a) antibodythat is specific for the CD33 antigen found on Acute Myeloid Leukemia(AML) cells (Roy et al. Blood 77:2404-2412 (1991)) and can be used totreat AML patients. Similarly, the monoclonal antibody anti-B4 is amurine IgG₁, that binds to the CD19 antigen on B cells (Nadler et al, J.Immunol. 131:244-250 (1983)) and can be used if the target cells are Bcells or diseased cells that express this antigen such as innon-Hodgkin's lymphoma or chronic lymphoblastic leukemia. Similarly, theantibody N901 is a murine monoclonal IgG₁ antibody that binds to CD56found on small cell lung carcinoma cells and on cells of other tumors ofneuroendocrine origin (Roy et al. J. Nat. Cancer Inst. 88:1136-1145(1996)), huC242 antibody that binds to the CanAg antigen, Trastuzumabthat binds to HER2/neu, and anti-EGF receptor antibody that binds to EGFreceptor.

Purification Methods

The conjugate, i.e., the finalized product, of the present invention ispurified to remove any unreacted or unconjugated effector or reportermolecule, or unreacted linker or unconjugated, hydrolyzed linker. Thepurification method can be a tangential flow filtration (TFF, also knownas cross flow filtration, ultrafiltration, or diafiltration), gelfiltration, adsorptive chromatography, selective precipitation, orcombinations thereof. The adsorptive chromatography methods includeion-exchange chromatography, hydroxyapatite chromatography, hydrophobicinteraction chromatography (HIC), hydrophobic charge inductionchromatography (HCIC), mixed mode ion exchange chromatography,immobilized metal affinity chromatography (IMAC), dye ligandchromatography, affinity chromatography, and reversed phasechromatography. For example, the conjugate Ab-(X′-L-Y′—C)_(m) describedin formula 2 is purified from unreacted C or unreacted/hydrolyzed linkerX-L-Y or X-L-Y′—C. Similarly, the conjugates described in formulae 4, 6,and 9 are purified. Such methods of purification are known to one ofskill in the art and can be found, for example, in US Publication No.2007/0048314.

Undesired Hydrolyzed Linker or Protein Cross-Linking in Conjugates

Traditional conjugation methods employing the initial reaction of aprotein with a heterobifunctional linker with reactive maleimide orhaloacetamide residue suffer from two major drawbacks: (i) the conjugateproduct may consist of hydrolyzed linker, due to aqueous inactivation ofthe incorporated linker in the antibody before reaction with theeffector or reporter molecule; and (ii) inter-or intrachaincross-linking of conjugate, due to reaction of maleimide (orhaloacetamide) group with the native histidine, lysine, tyrosine, orcysteine residues in protein or peptide (A. Papini et al., Int. J. Pept.Protein Res., 1992, 39, 348-355; T. Ueda et al., Biochemistry, 1985, 24,6316-6322). Such interchain cross-linking in antibody would result invarious non-reducible covalent linkages between the heavy and lightchains, or between two heavy chains, which would be apparent in reducingSDS-PAGE analysis as bands of higher molecular weights than the expectedheavy and light chain bands. Such interchain or intrachain cross-linkingin antibody would also be apparent by MS as peaks of aberrant massesdifferent than the expected masses of antibody plus linked reporter oreffector groups. Unlike traditional conjugation methods, the methoddescribed in this application results in conjugates with highhomogeneity with no substantial interchain cross-linking or hydrolyzedlinker.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXAMPLES

The following examples, which are illustrative only, are not intended tolimit the present invention.

Example 1 Conjugation of Antibody with Cytotoxic Agent DM1/DM4 UsingHeterobifunctional Linker Maleimide-PEG_(n)-NHS by this Method (FIG. 1)versus Traditional Two-Step Method

Stock solutions of DM1[N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine], or DM4[N^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl)maytansine](DMx) thiol and the Maleimide-PEG_(n)-NHS bifunctional linker were madeup in N,N-dimethylacetamide (DMA) at concentrations of 30-60 mM. Thelinker and DMx thiol were mixed together in DMA containing up to 50% v/vof 200 mM succinate buffer, 2 mM EDTA, pH 5.0 to give a molar ratio ofDMx to linker of 1.6:1 and a final concentration of DMx equal to 15 mM.After mixing, the reaction mixture was left for 1-4 h and then analiquot of the reaction mixture was diluted 10 fold and its absorbancemeasured at 302-320 nm to determine the presence of any remainingunreacted maleimide using the extinction coefficient (ε) of maleimide at302 nm=620 M⁻¹ cm⁻¹, and ε320 nm˜450 M⁻¹ cm⁻¹. (Additional reverse phaseHPLC analysis of a frozen aliquot of the reaction mixture was carriedout later with absorbance monitoring at 302 nm and 252 nm to verifycomplete disappearance of linker maleimide and formation of the desiredlinker-DMx reagent at the time of addition of the reaction mixture toantibody). When no further maleimide was present by UV, an aliquot ofthe reaction mixture was added without purification to a solution ofantibody in phosphate buffer (pH 7.5) under final conjugation conditionsof 4 mg/ml Ab, 90% phosphate buffer/10% DMA, pH 7.5. The conjugationreaction was allowed to proceed at ambient temperature for 2 h. Ab-DMxconjugate was purified from the excess small-molecule DMx and linkerreactants using a G25 gel filtration column equilibrated in pH 7.5phosphate buffer, or using tangential flow filtration (TFF). Theconjugation mixture was further kept at 4° C. for 2 days in pH 7.5buffer to allow the dissociation of any DMx species attached to antibodynon-covalently or via labile linkage. The conjugate was then dialyzedovernight into pH 5.5 histidine/glycine buffer and then filtered througha 0.22 μm filter for final storage. The number of DMx molecule per Abmolecule (average) in the final conjugate was measured by determiningabsorbance of the conjugate at 252 and 280 nm and using known extinctioncoefficients for DMx and antibody at these two wavelengths.

Several different reaction conditions were used for the initial reactionof DMx thiol with the heterobifunctional maleimide-PEG₄-NHS reagent: 50%DMA/50% aqueous 200 mM succinate buffer pH 5.0, 2 mM EDTA (v/v); or 60%DMA/40% 200 mM succinate buffer pH 5.0, 2 mM EDTA (v/v); or 100% DMAwith 1.5 molar equivalents of an organic base (for exampleN,N′-diisopropyl ethylamine, DIPEA, or 4-methylmorpholine) per mol DM4thiol.

In one series of experiments, the molar equivalent of DMx tomaleimide-PEG₄-NHS linker (purchased from Pierce Endogen) was variedfrom 1.2-2.4, and the reaction time was 30 min. The number of DMx/Abmeasured on purified conjugates were measured as a function of addedequivalents of DMx per linker. Conditions of 1.2-2.0 equivalents ofDM1/Linker gave conjugates with similar DMx/Ab loads, indicating thatthe undesired reaction of the DMx thiol at the NHS ester side of thelinker is not a significant problem. The amount of cross-linking presentin the final conjugates was also analyzed by reducing SDS PAGE showingthat the presence of cross-linked contaminants decreases significantlywith increasing DM1/linker ratio.

One optional quenching step using maleimide or haloacetamide reagents(such as maleimidobutyric acid, or maleimidopropionic acid, orN-ethylmaleimide, or iodoacetamide, or iodoacetic acid) was introducedafter the completion of the initial DMx and heterobifunctional linkerreaction (before the addition of the reaction mixture to the antibody)to quench the excess DMx thiol group in order to prevent any unwantedreaction of DMx thiol with the antibody.

An alternate method of the reaction of antibody with the unpurifiedinitial reaction mixture of DMx and heterobifunctional linker involvedmixing the initial reaction mixture of DMx and heterobifunctional linker(upon completion of the DMx-linker reaction) with antibody at low pH (pH˜5) followed by the addition of buffer or base to increase the pH to6.5-8.5 for the conjugation reaction.

An antibody-PEG₄-Mal-DM1 or DM4 conjugate was made by the traditionaltwo step conjugation method for comparison with the conjugation methoddescribed in this invention. The humanized antibody at a concentrationof 8 mg/ml in pH 7.5 phosphate buffer (50 mM potassium phosphate, 50 mMsodium chloride, 2 mM EDTA, pH 7.5) and 5% DMA was modified with excessheterobifunctional maleimide-PEG₄-NHS linker reagent (purchased fromPierce Endogen). After 2 h at 25° C., the modified antibody was gelpurified by G25 chromatography to remove excess unreacted,unincorporated linker. The recovery of purified Ab was determined by UVabsorbance at 280 nm. The number of linked maleimide groups in themodified Ab was determined using a small aliquot of modified antibody byaddition of a known amount of thiol (such as 2-mercaptoethanol), addedin excess over the maleimide, to react with the maleimide residues inthe modified antibody and then assaying the remaining thiol by Ellman'sassay using DTNB reagent (extinction coefficient of TNB thiolate at 412nm=14150 M⁻¹ cm⁻¹; Riddles, P. W. et al., Methods Enzymol., 1983, 91,49-60; Singh, R., Bioconjugate Chem., 1994, 5, 348-351). The conjugationof modified Ab with DM1 or DM4 thiol was carried out at an Abconcentration of 2.5 mg/ml in a reaction mixture consisting of 95%phosphate buffer pH 7.5 (50 mM potassium phosphate, 50 mM sodiumchloride, 2 mM EDTA, pH 7.5) and 5% DMA. An excess of 1.7 molarequivalents of DM1 or DM4 thiol was added per mol of linked maleimide onthe Ab. After reacting overnight at 25° C., the conjugate was sterilefiltered using a 0.22 μm filter and gel purified from excess unreactedDM1 or DM4 by a G25 column equilibrated in phosphate buffer pH 7.5 (50mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA, pH 7.5). Thepurified conjugate was held at 4° C. for 2 days in phosphate buffer pH7.5 (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA, pH7.5) to allow for the dissociation of any DM1 or DM4 species attached toantibody non-covalently or via a labile linkage. The conjugate wassubsequently dialyzed for 2 days in histidine/glycine buffer pH 5.5 (130mM glycine/10 mM histidine, pH 5.5) and sterile filtered using a 0.22 μmfilter. The number of DM1 or DM4 molecules per Ab molecule in the finalconjugate was measured by determining absorbance of the conjugate at 252and 280 nm using known extinction coefficients for DM1/DM4 and Ab atthese two wavelengths.

Reducing SDS PAGE was carried out on conjugate and antibody samplesusing the NuPage electrophoresis system with a 4-12% Bis Tris Gel(Invitrogen). Heat denatured and reduced samples were loaded at 10μg/lane. The reducing SDS-PAGE of the conjugates prepared using themethod described in this invention showed only the expected heavy andlight chain bands (50 kDa and 25 kDa respectively) as the major bands(FIG. 2). In contrast, the conjugates prepared by the traditionaltwo-step conjugation method showed undesired cross-linked bands withmolecular weights of 75, 100, 125, and 150 kDa, presumably correspondingto inter-chain cross-linked species HL, H₂, H₂L, and H₂L₂ respectively(FIG. 2).

Protein LabChip electrophoresis analysis (under reducing condition) ofthe antibody-PEG₄-Mal-DM4 conjugate prepared by the method described inthis invention showed the expected heavy and light chain bands withpercentages of 58 and 30% (of total protein), which are similar to thosefor unconjugated antibody of 65 and 30% respectively (FIGS. 3A-B). Incontrast, the conjugate prepared using the traditional two-stepconjugation method showed heavy and light bands of only 16 and 8%respectively, and major bands of higher molecular weights ranging from94-169 kDa presumably due to inter-chain cross-linking (FIGS. 3A-B).Based on the quantitative Protein LabChip analysis, the conjugateprepared by the method described in this application is highly superiorto that prepared using the conventional two-step process.

The MS analysis of the conjugates prepared by the method described inthis invention showed discrete DMx-antibody conjugate peaks for antibodybearing increasing numbers of maytansinoid molecules per antibodymolecule (FIG. 4B). In contrast, the MS of the conjugate obtained usingthe traditional 2-step method was nearly unresolved suggestinginhomogeneity of the conjugate preparation presumably due tocross-linking or inactivated maleimide linker (FIG. 4A). Based on MS,therefore, the conjugate prepared using the method described in thisinvention is superior to that synthesized by the traditional two-stepmethod.

The binding of an anti-CanAg Ab-PEG₄-Mal-DM1 conjugate prepared by themethod described in this invention was measured by flow cytometry usingthe antigen-expressing COLO205 cells, and was found to be similar tothat of unconjugated antibody suggesting that the conjugation had nodetrimental effect on the binding of the antibody (FIG. 5). Thecytotoxic activity of the anti-CanAg Ab-PEG₄-Mal-DM1 conjugate preparedby the method described in this invention was measured in vitro usingCOLO205 colon cancer cells expressing the CanAg antigen (FIG. 6). Theantigen-expressing cancer cells were plated at around 1000 cells/well ina 96 well plate in cell culture media containing fetal bovine serum andexposed to varying concentrations of Ab-DMx conjugate. After a 5 dayexposure to the conjugate, the viable cells remaining in each well weremeasured using the WST-8 assay (Dojindo Molecular Technologies). Asshown in FIG. 6, the anti-CanAg Ab-PEG₄-Mal-DM1 conjugate prepared usingthis method was highly potent at low concentrations toward CanAgantigen-expressing COLO205 colon cancer cells. The cytotoxicity of theanti-CanAg Ab-PEG₄-Mal-DM1 conjugate prepared by the method described inthis invention was specific to COLO205 cells as it could be blocked bythe addition of excess, unconjugated antibody.

Example 2 Conjugation of Antibody with DM1/DM4 Using Maleimide-Sulfo-NHSLinker by this Method (FIG. 7) versus Traditional Sequential Two-StepMethod

Stock solutions of DMx thiol and the maleimide-Sulfo-NHSheterobifunctional linker were made up in N,N-dimethylacetamide (DMA) atconcentrations of 30-60 mM. The linker and DMx thiol were mixed togetherin DMA containing up to 40% v/v of 200 mM succinate buffer, 2 mM EDTA,pH 5.0 to give a ratio of DMx to linker of 1.6 and a final concentrationof DMx equal to 15 mM. After mixing, the reaction was left for 1-4 h andthen an aliquot of the reaction mixture was diluted 10 fold to measurethe absorbance at 302-320 nm for assessing the completion of reactionand the absence of maleimide. (Additional reverse phase HPLC analysis ofa frozen aliquot of the reaction mixture was carried out later withabsorbance monitoring at 302 nm and 252 nm to verify completedisappearance of linker maleimide and formation of the desiredlinker-DMx reagent at the time of addition of the reaction mixture toantibody). When no further maleimide was present by UV, an aliquot ofthe reaction mixture was added to a mixture of antibody in phosphatebuffer (pH 7.5) under final conjugation conditions of 4 mg/ml Ab, 90%phosphate buffer/10% DMA, pH 7.5. The conjugation reaction was allowedto proceed at ambient temperature for 2 h. The Ab-DMx conjugate waspurified from excess unreacted DMx and unconjugated linker productsusing a G25 gel filtration column equilibrated in pH 7.5 phosphatebuffer or by tangential flow filtration. The conjugate was kept at 4° C.for 2 days in pH 7.5 buffer to allow the dissociation of any DMx speciesattached to antibody non-covalently or via labile linkage. The conjugatewas then dialyzed overnight into pH 5.5 histidine/glycine buffer andthen filtered through a 0.22 μm filter for final storage. The number ofDMx molecules per Ab antibody molecule (average) in the final conjugatewas measured by determining absorbance of the conjugate at 252 and 280nm and using known extinction coefficients for DMx and antibody at thesetwo wavelengths.

For comparison, the Ab-Sulfo-Mal-DMx conjugates were prepared using thetraditional 2-step conjugation method. The antibody (Ab) at aconcentration of 8 mg/ml in pH 7.5 phosphate buffer/5% DMA buffer wasmodified with excess bifunctional maleimide-Sulfo-NHS linker. Thereaction was allowed to proceed at 20° C. for 2 h and then the modifiedAb was purified away from excess unreacted linker using G25chromatography. The recovery of purified Ab was determined by UVabsorbance at 280 nm. The number of linked maleimide groups in themodified Ab was determined using a small aliquot of modified antibody byaddition of a known amount of thiol (such as 2-mercaptoethanol), addedin excess over the maleimide, to react with the maleimide residues inthe modified antibody and then assaying the remaining thiol by Ellman'sassay using DTNB reagent (extinction coefficient of TNB thiolate at 412nm=14150 M⁻¹ cm⁻¹; Riddles, P. W. et al., Methods Enzymol., 1983, 91,49-60; Singh, R., Bioconjugate Chem., 1994, 5, 348-351). The conjugationof modified Ab with DMx was carried out at an antibody concentration of2.5 mg/ml in 95% pH 7.5 phosphate buffer/5% DMA (v/v), with 1.7 molarequivalents of DMx thiol added per mol of linked maleimide in the Ab.The reaction was left for 8-24 h at 18° C. and the conjugate wasseparated from excess, unreacted DMx via G25 size-exclusionchromatography. After purification the conjugate was kept at 4° C. for 2days in pH 7.5 buffer to allow the dissociation of any DMx speciesattached to antibody non-covalently or via labile linkage. The conjugatewas then dialyzed overnight into pH 5.5 histidine/glycine buffer andthen filtered through a 0.22 μm filter for final storage. The number ofDMx molecule per Ab molecule in the final conjugate was measured bydetermining absorbance of the conjugate at 252 and 280 nm and usingknown extinction coefficients for DMx and antibody at these twowavelengths.

Reducing SDS PAGE was carried out using conjugate and antibody samples(10 μg/lane) using the NuPage electrophoresis system (Invitrogen) with aNuPage 4-12% Bis Tris Mini Gel and NuPAGE MOPS SDS running buffer (FIG.8). Bands on the gel with molecular weights of 75, 125, and 150 kDa wereindicative of inter-chain cross-linked species (HL, H₂L and H₂L₂respectively). A comparison of Ab-Sulfo-Mal-DM1 conjugates with ˜4DM1/Ab (lane 3, by this method, and lane 2, by traditional 2-stepconjugation method, respectively) and ˜6 DM1/Ab (lane 5, by this method,and lane 4, by traditional 2-step conjugation method, respectively)clearly shows that conjugates made via the method described in thisinvention (lanes 3 and 5) have much smaller proportion of high molecularweight cross-linked species than conjugates made by the traditional2-step method (lanes 2 and 4).

Protein LabChip electrophoresis analysis (under reducing condition) ofthe antibody-Sulfo-Mal-DM1 conjugate prepared by the method described inthis invention showed the heavy and light chain major bands withpercentages of 70 and 28% (of total protein), which are similar to thosefor unconjugated antibody of 70 and 30% respectively (FIGS. 9A-B). Incontrast, the conjugate prepared using the traditional two-step methodshowed heavy and light bands of only 53 and 23% respectively, and majorbands of higher molecular weights ranging from 99-152 kDa presumably dueto inter-chain cross-linking (FIGS. 9A-B). Based on the quantitativeProtein LabChip analysis, the conjugate prepared by the method describedin this application is much superior in terms of lack of inter-chaincross-linking compared to that prepared using the conventional two-stepprocess.

The Ab-Sulfo-mal-DM1 conjugates with similar drug loads made via themethod described in this invention and by the traditional two stepmethod were compared by size exclusion LC/MS analysis (FIGS. 10A-B). Theconjugates made via the method described in this invention show thedesired MS spectrum containing only the expected distribution of peakswith mass equal to Ab-(linker-DMx)_(n) (FIG. 10A). In the case ofconjugates made using the traditional two-step method, the major peaksin the spectra all contain one or more hydrolyzed or cross-linked linkerfragments in addition to the desired Ab-(linker-DMx)_(n) moieties (FIG.10B). The putative mechanism of the inter-chain cross-linking or aqueousinactivation of maleimide in the traditional 2-step reaction sequence isshown in FIG. 17, whereby the incorporated maleimide (or haloacetamide)residue from the initial reaction of antibody with theheterobifunctional linker can react with intramolecular (orintermolecular) histidine, lysine, tyrosine, or cysteine residuesresulting in inter-chain cross-linking, or the initially incorporatedmaleimide (or haloacetamide) residue can get inactivated (such as byhydrolytic maleimide ring cleavage or by aqueous addition to maleimide)and therefore become unavailable for the rapid reaction withthiol-bearing effector or reporter group. Thus the LC-MS analysisclearly shows that the method described in this invention has theadvantage of producing homogeneous conjugate with little or nohydrolyzed or cross-linked linker fragments attached to antibody.

The binding of an anti-CanAg Ab-Sulfo-Mal-DM1 conjugate with 5.6maytansinoid load per antibody molecule (average) prepared by the methoddescribed in this invention was measured by flow cytometry using theantigen-expressing COLO205 cells, and was found to be similar to that ofunconjugated antibody suggesting that the conjugation did not affect thebinding of the antibody to target antigen (FIG. 11). The cytotoxicactivity of the anti-CanAg Ab-Sulfo-Mal-DM1 conjugate prepared by themethod described in this invention was measured in vitro using COLO205colon cancer cells expressing the CanAg antigen (FIG. 12). Theantigen-expressing cancer cells were plated at around 1000 cells/well ina 96 well plate in cell culture media containing fetal bovine serum andexposed to varying concentrations of Ab-DMx conjugate. After a 5 dayexposure to the conjugate, the viable cells remaining were measuredusing the WST-8 assay (Dojindo Molecular Technologies). As shown in FIG.12, the anti-CanAg Ab-Sulfo-Mal-DM1 conjugate prepared using this methodwas highly potent at low concentrations toward CanAg antigen-expressingCOLO205 colon cancer cells. The cytotoxicity of this conjugate wasspecific as it could be blocked by competition with excess, unconjugatedantibody.

An alternative method of conjugation using the method described in thisinvention involved a quenching step using maleimide or haloacetamidereagents (such as 4-maleimidobutyric acid, or 3-maleimidopropionic acid,or N-ethylmaleimide, or iodoacetamide, or iodoacetic acid) after thecompletion of the initial DMx and heterobifunctional linker reaction(before the addition of the reaction mixture to the antibody) to quenchthe excess DMx thiol group in order to prevent any unwanted reaction ofDMx thiol with the antibody. In a specific example, following completionof the initial DMx and heterobifunctional linker reaction (before theaddition of the reaction mixture to the antibody), 4-maleimidobutyricacid was added to quench the excess DMx thiol group in order to preventany unwanted reaction of DMx thiol with the antibody during theconjugation reaction. To a reaction mixture of DM4 and Sulfo-Mal-NHSheterobifunctional reagent that contained an excess of DM4 (3 mM), uponcompletion of the desired DM4 thiol coupling to the maleimide group ofthe heterobifunctional reagent, a two-fold molar excess of4-maleimidobutyric acid (6 mM) was added to the reaction mixture atambient temperature for 20 minutes to quench the remaining DM4 from theinitial coupling reaction. Without purification of the reaction mixture,an aliquot was mixed with a solution of antibody in phosphate buffer (pH7.5) under final conjugation conditions of 4 mg/ml Ab, 90% aqueousphosphate buffer/10% DMA, pH 7.5. The conjugation reaction was allowedto proceed at ambient temperature for 2 h. The antibody-DM4 conjugatewas purified from the excess small-molecule DM4 and linker reactantsusing a G25 gel filtration column equilibrated in pH 7.5 phosphatebuffer. The conjugation mixture was further kept at 4° C. for 2 days inpH 7.5 buffer to allow the dissociation of any DMx species attached toantibody non-covalently or via labile linkage. The conjugate was thendialyzed overnight into pH 5.5 histidine/glycine buffer and filteredthrough a 0.22 μm filter for final storage. The average number of DM4molecules per Ab molecule in the final conjugate was measured bydetermining absorbance of the conjugate at 252 and 280 nm and usingknown extinction coefficients for DM4 and antibody at these twowavelengths. The conjugate samples were analyzed by non-reducing SDSPAGE using the NuPage electrophoresis system with a 4-12% Bis Tris Gel(Invitrogen). The heat-denatured samples were loaded at 10 μg/lane. Thenon-reducing SDS-PAGE of the conjugate prepared using the methoddescribed in this invention (without quenching) showed evidence of alight chain band (˜25 kDa) and half-antibody band (heavy-light chain;˜75 kDa) (FIG. 18). On the other hand, the conjugate prepared using themethod described in this invention which was treated with4-maleimidobutyric acid (to cap excess DMx thiol) had significantlylower amounts of these undesirable bands (at levels comparable tounmodified antibody sample). Another advantage of the quenching of theinitial DMx and heterobifunctional reaction mixture (before conjugationwith the antibody) by thiol-quenching reagents such as4-maleimidobutyric acid is that during the antibody conjugation reactionthere is no “free” DMx (DM1 or DM4) species and therefore the finalconjugate after purification does not contain “free” or unconjugated DMxspecies. The DMx-adduct with 4-maleimidobutyric acid (or other polarthiol-quencing reagents) is more water soluble than DMx and thereforecan be more easily separated from the covalently linked antibody-DMxconjugate.

Example 3 Conjugation of Antibody with Maytansinoid (DM1/DM4) Usingsulfo-NHS-SMCC Linker (FIG. 13)

Stock solutions of DM1 or DM4 thiol (DMx) and the sulfo-SMCCheterobifunctional linker with sulfo-NHS group (purchased from PierceEndogen; FIG. 13) were prepared in DMA at concentrations of 30-60 mM.Linker and DM1 or DM4 thiol were mixed together in DMA containing up to40% v/v of aqueous 200 mM succinate buffer, 2 mM EDTA, pH 5.0 to give aratio of DM1 or DM4 (DMx) to linker of 1.6:1 and a final concentrationof DMx of 6 mM. After mixing, the reaction was left for 1-4 h at ambienttemperature and then an aliquot of the reaction mixture was diluted10-fold to measure absorbance at 302-320 nm to assess whether all of themaleimide had reacted. (Additional reverse phase HPLC analysis of afrozen aliquot of the reaction mixture was carried out later withmonitoring at 302 nm and 252 nm to verify complete disappearance oflinker maleimide and formation of the desired sulfo-NHS-linker-Mal-DMxreagent at the time of addition of the reaction mixture to antibody).When no further maleimide was present by UV an aliquot of the reactionwas added to an aqueous solution of an antibody in phosphate buffer (pH7.5) under final conjugation conditions of 4 mg/ml Ab, 90% phosphatebuffer (aqueous)/10% DMA (v/v), pH 7.5. The conjugation reaction wasallowed to proceed at ambient temperature for 2 h. Ab-DMx conjugate waspurified from excess unreacted reagent and excess DMx using a G25 gelfiltration column equilibrated in pH 7.5 phosphate buffer (aqueous).Conjugate was kept at 4° C. for 2 days in pH 7.5 buffer to allow thedissociation of any DMx species attached to Ab non-covalently or vialabile linkage. The conjugate was then dialyzed overnight into pH 5.5histidine/glycine buffer and then filtered through a 0.22 μm filter forfinal storage. The number of DMx molecule per Ab molecule in the finalconjugate was measured by determining absorbance of the conjugate at 252and 280 nm and using known extinction coefficients for DMx and antibodyat these two wavelengths.

For comparison, the Ab-SMCC-DMx conjugates were prepared using thetraditional 2-step conjugation method. The antibody (Ab) at aconcentration of 8 mg/ml in 95% pH 6.5 phosphate buffer/5% DMA bufferwas modified with excess bifunctional sulfo-SMCC linker with sulfo-NHSgroup (purchased from Pierce Endogen). The reaction was allowed toproceed at 25° C. for 2 h and then the modified Ab was purified awayfrom excess unreacted linker using G25 chromatography. The recovery ofpurified Ab was determined by UV absorbance at 280 nm. The number oflinked maleimide groups in the modified Ab was determined using a smallaliquot of modified antibody by addition of a known amount of thiol(such as 2-mercaptoethanol), added in excess over the maleimide, toreact with the maleimide residues in the modified antibody and thenassaying the remaining thiol by Ellman's assay using DTNB reagent(extinction coefficient of TNB thiolate at 412 nm=14150 M⁻¹ cm⁻¹;Riddles, P. W. et al., Methods Enzymol., 1983, 91, 49-60; Singh, R.,Bioconjugate Chem., 1994, 5, 348-351). The conjugation of modified Abwith DM1 or DM4 was carried out at an antibody concentration of 2.5mg/ml in 95% pH 6.5 phosphate buffer/5% DMA (v/v), with 1.7 molarequivalents of DM1 or DM4 thiol added per mol of linked maleimide in theAb. The reaction was left for 8-24 h at 18° C. and the conjugate wasseparated from excess, unreacted DM1 (or DM4) via G25 chromatography.After purification the conjugate was kept at 4° C. for 2 days in pH 6.5buffer to allow the hydrolysis of any weakly linked DM1/DM4 species. Theconjugate was then dialyzed overnight into pH 5.5 histidine/glycinebuffer and then filtered through a 0.22 μm filter for final storage. Thenumber of DM1/DM4 molecules per Ab molecule in the final conjugate wasmeasured by determining absorbance of the conjugate at 252 and 280 nmand using known extinction coefficients for DM1/DM4 and antibody atthese two wavelengths.

Reducing SDS PAGE was carried out on conjugate and antibody samples (10μg/lane) using the NuPage electrophoresis system (Invitrogen) with aNuPage 4-12% Bis Tris Mini Gel and NuPAGE MOPS SDS running buffer (FIG.14). Bands on the gel with molecular weights of 75, 125, and 150 kDawere indicative of inter-chain cross-linked species (HL, H₂L and H₂L₂respectively). A comparison of Ab-SMCC-DM1 conjugates with 3.1 D/Ab(lane 4, by this method, and lane 3, by the traditional 2-step method,respectively) clearly shows that conjugate made via the method describedin this invention (lane 4) has much fewer high molecular weightcross-linked species than conjugates made by the traditional 2 stepmethod (lane 3).

Protein LabChip electrophoresis analysis (under reducing condition) ofthe antibody-SMCC-DM1 conjugate prepared by the method described in thisinvention showed the heavy and light chain major bands with percentagesof 67 and 30% (of total protein), which are similar to those forunconjugated antibody of 68 and 30% respectively (FIGS. 15A-B). Incontrast, the conjugate prepared using the traditional two-step methodshowed heavy and light bands of only 54 and 24% respectively, and majorbands of higher molecular weights ranging from 96-148 kDa presumably dueto inter-chain cross-linking (FIGS. 15A-B). Based on the quantitativeProtein LabChip analysis, the conjugate prepared by the method describedin this application is much superior in terms of lack of inter-chaincross-linking compared to that prepared using the conventional two-stepprocess.

The Ab-SMCC-DM1 conjugates with similar drug loads made via the methoddescribed in this invention and by the traditional two step method werecompared by size exclusion LC/MS analysis (FIGS. 16A-B). The conjugatemade via the method described in this invention shows the desired MSspectrum containing only the expected distribution of peaks with massequal to Ab-(linker-DMx)_(n) (FIG. 16B). In the case of conjugate madeusing the traditional two-step method the spectrum shows a heterogeneousmixture of species which includes the desired Ab-(linker-DMx)_(n)species plus additional species containing inactivated maleimide andcross-linked linker fragments (FIG. 16A). The putative mechanisms of theinter-chain cross-linking and maleimide inactivation in the traditional2-step reaction sequence are shown in FIG. 17 whereby the incorporatedmaleimide (or haloacetamide residue) from the initial reaction ofantibody with the heterobifunctional linker can react withintramolecular (or intermolecular) histidine, lysine, tyrosine, orcysteine residues resulting in inter-chain cross-linking, or theinitially incorporated maleimide (or haloacetamide) residue can getinactivated by hydrolysis or hydration of the maleimide residue beforethe reaction step with the thiol-bearing DM1 or DM4 (DMx) agent. Thusthe LC-MS analysis clearly shows that the method described in thisinvention has the advantage of producing homogeneous conjugate withlittle or no inactivated maleimide or cross-linked linker fragmentsattached to antibody.

Example 4 Conjugation of Antibody with DM1/DM4 (DMx) with Cleavable,Disulfide Linkers by this Method (FIG. 19)

Stock solutions containing DM1 or DM4 thiol (DMx) and heterobifunctionallinker 4-(2-pyridyldithio)butanoic acid-N-hydroxysuccinimide ester(SPDB) were prepared in DMA at concentrations of 30-60 mM. Linker andDMx thiol were mixed together in DMA containing up to 40% v/v of aqueous200 mM succinate buffer, 2 mM EDTA, pH 5.0 to give a ratio of DM1 or DM4(DMx) to linker of 1.6:1 and a final concentration of DMx of 8 mM. Aftermixing, the reaction was left for 1 h at ambient temperature and then analiquot of the reaction was added to an aqueous solution of antibody inphosphate buffer (pH 7.5) under final conjugation conditions of 4 mg/mlAb, 90% phosphate buffer (aqueous)/10% DMA (v/v), pH 7.5. Theconjugation reaction was allowed to proceed at ambient temperature for 2h. The Ab-DMx conjugate was purified from excess unreacted reagent andexcess DMx using a G25 gel filtration column equilibrated in pH 7.5phosphate buffer (aqueous). Conjugate was kept at 4° C. for 2 days in pH7.5 buffer to allow for the dissociation of any DMx species attached toAb non-covalently or via labile linkage. The conjugate was then dialyzedovernight into pH 5.5 histidine/glycine buffer and then filtered througha 0.22 μm filter for final storage. The number of DMx molecules per Abmolecule on the final conjugate was measured by determining absorbanceof the conjugate at 252 and 280 nm using known extinction coefficientsfor DMx and antibody at these two wavelengths.

Example 5 Preparation of Antibody-DM1/DM4 (Ab-DMx) Conjugate with BothDisulfide- and Non-Cleavable Linkers Using this Method (FIG. 20)

Stock solutions of DM1 or DM4 thiol (DMx) and the NHS-PEG_(n)-Maleimideheterobifunctional linker were prepared in N,N-dimethylacetamide (DMA)at concentrations of 30-80 mM. The NHS-PEG₄-Maleimide linker and DMxthiol were mixed together in DMA containing up to 40% v/v of 200 mMsuccinate buffer, 2 mM EDTA, pH 5.0 to give a molar ratio of DMx tolinker of 1.6:1 and a final concentration of DMx equal to 8.0 mM. Thereaction mixture was left to react for 2 h at ambient temperature. In aseparate parallel reaction, SPDB linker and DMx thiol were mixedtogether and reacted in a similar fashion to the conditions used forNHS-PEG₄-maleimide reaction except for a reaction time of 1 h. After thecompletion of both reactions and without purification, equal volumes ofPEG₄-Mal-DM4 mixture and SPDB-DM4 mixture were combined. An aliquot ofthe combined reaction mixtures was added without purification to asolution of antibody in phosphate buffer (pH 7.5) under finalconjugation conditions of 4 mg/ml Ab, 90% phosphate buffer (aqueous)/10%DMA (v/v), pH 7.5. The conjugation reaction was allowed to proceed atambient temperature for 2 h. Ab-DMx conjugate was purified from excessunreacted reagents and excess DMx using a G25 gel filtration columnequilibrated in pH 7.5 phosphate buffer (aqueous). The conjugate waskept at 4° C. for 2 days in pH 7.5 buffer to allow for the dissociationof DMx species attached to Ab non-covalently or via labile linkage. Theconjugate was then dialyzed overnight into pH 5.5 histidine/glycinebuffer and then filtered through a 0.22 μm filter for final storage. Thenumber of DMx molecules per Ab molecule on the final conjugate wasmeasured by determining absorbance of the conjugate at 252 and 280 nmusing known extinction coefficients for DMx and antibody at these twowavelengths.

The Ab-(mixed SPDB and PEG₄-Mal linker)-DMx conjugate made via themethod described in this invention was tested to determine the percentof incorporation of cleavable versus non-cleavable linker on the Ab bycomparing DMx per antibody (D/A) ratio before and after DTT(dithiothreitol) treatment of the conjugate to reduce the disulfidelinkage. In order to maintain reaction pH at 7.5 during DTT reduction,the conjugate was first dialyzed into 250 mM HEPES buffer pH 7.5. Theconjugate was then reduced by reacting with 25 mM DTT for 20 min at 37°C. After the DTT reaction, the released DMx and DTT were separated fromthe reaction mixture using a G25 gel filtration column equilibrated in250 mM HEPES buffer pH 7.5. The average number of DMx molecules per Abmolecule in the purified product was measured by determining theabsorbance of the conjugate at 252 and 280 nm and using known extinctioncoefficients for DMx and antibody at these two wavelengths. The ratiobetween D/A of DTT-treated conjugate and D/A of non-DTT treatedconjugate was used to calculate the percent of DMx attached to Ab vianon-cleavable linkage. Two additional samples, Ab-SPDB-DM4 andAb-PEG₄-Mal-DM4 conjugates, were treated with DTT as positive andnegative controls, respectively. By comparing D/A ratio before and afterDTT treatment, the control non-cleavable Ab-PEG₄-Mal-DM4 conjugateshowed that approximately all linkers bound were found to benon-cleavable (93%) as expected. The Ab-(mixed SPDB and PEG₄Mallinker)-DMx conjugate containing both non-cleavable and disulfidelinkers made via the method described in this invention had 41% less DMxcleaved by DTT treatment relative to the amount of DMx loss from theAb-SPDB-DMx conjugate that consists entirely of cleavable linker. Thisdemonstrated that the Ab-(mixed SPDB and PEG₄-Mal)-DMx conjugate madevia the method described in this invention was composed of approximately40% non-cleavable and 60% cleavable linkers. By changing the initialratio of the non-cleavable and cleavable linker reagents, conjugates ofantibody with maytansinoid or other effector can be prepared withdifferent ratio of non-cleavable and cleavable linkers. FIG. 21 showsthe mass spectrum of deglycosylated conjugate described above, whichcomprises of antibody with an average of 3.5 maytansinoid molecules perantibody molecule linked via both disulfide linkers (SPDB) andnon-cleavable linkers (PEG). The MS shows discrete conjugate speciesbearing both cleavable and non-cleavable linkers (FIG. 21). For example,the conjugate peak designated D2-PEG-SPDB bears one disulfide-linked andone non-cleavable thioether-linked maytansinoid molecule; the conjugatepeak designated D3-PEG-2SPDB bears two disulfide-linked and onenon-cleavable thioether-linked maytansinoid molecules; and the conjugatepeak designated D3-2PEG-SPDB bears one disulfide-linked and twonon-cleavable thioether-linked maytansinoid molecules.

Example 6 Conjugation of Antibody with Maytansinoid Using SMCC Linker(FIG. 22)

Stock solutions of DM1 thiol and SMCC heterobifunctional linker (Pierce)were prepared in DMA at concentrations of 30-60 mM. Linker and DM1 thiolwere mixed together in DMA containing up to 50% v/v of aqueous 200 mMsuccinate buffer, 2 mM EDTA, pH 5.0 to give a ratio of DM1 to linker of1.4:1 mole equivalent and a final concentration of DM1 of 1 to 6 mM.After mixing, the reaction was left for up to 4 h at ambient temperatureand then an aliquot of the reaction mixture was diluted 10-fold tomeasure absorbance at 302-320 nm to assess whether all of the maleimidehad reacted with thiol. When no further maleimide was present by UV, analiquot of the reaction was added to an aqueous solution of an antibodyin phosphate buffer (pH 7.5-8.5) under final conjugation conditions of2.5 mg/ml Ab, 70-80% phosphate buffer (aqueous)/30-20% DMA (v/v). Theconjugation reaction was allowed to proceed at ambient temperature for 3h. Ab-DM1 conjugate was purified from excess unreacted or hydrolyzedreagent and excess DM1 using a G25 gel filtration column equilibrated inpH 7.4 phosphate buffer (aqueous). The conjugate was then dialyzedovernight into pH 7.4 phosphate buffer (aqueous) and then filteredthrough a 0.22 μm filter for final storage. The number of DM1 moleculeper Ab molecule in the final conjugate was measured by determiningabsorbance of the conjugate at 252 and 280 nm and using known extinctioncoefficients for DM1 and antibody at these two wavelengths. Similarly,conjugates of antibody with DM4 thiol and SMCC can be prepared. Theseconjugates of antibody with DM1 or DM4 using SMCC linker containthioether non-cleavable linker.

The Ab-SMCC-DM1 conjugate made via the method described in thisinvention was characterized by MS analysis of deglycosylated conjugate(FIG. 23). The conjugate made via the method described in this inventionshows the desired MS spectrum containing the expected distribution ofpeaks with mass equal to Ab-(linker-DM1)_(n).

Example 7 Conjugation of Antibody with Maytansinoid UsingHeterobifunctional Disulfide-Containing Linkers (SSNPB, SPP)

Disulfide containing heterobifunctional linkers SSNPB(N-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio)butyrate) and SPP(N-succinimidyl-3-(2-pyridyldithio)propionate) can be used to preparedisulfide-linked antibody-maytansinoid conjugates by the method similarto that described for SPDB linker in Example 4. The structure of thedisulfide-linked conjugate prepared using SPDB (FIG. 19) is identical tothat of the conjugate prepared with SSNPB (FIG. 24). The MS of adisulfide-linked conjugate prepared using SPDB showed discrete peakswith mass values corresponding to different numbers of maytansinoidmolecules attached to antibody.

Example 8 Conjugation of Antibody with Maytansinoid ContainingNon-Cleavable Linkers with Linear Alkyl Carbon Chain

Conjugates containing non-cleavable linker with linear alkyl carbonchain were prepared using reaction mixture of maytansinoid andheterobifunctional linkers with linear alkyl carbon chain, similar tothe method described for SMCC linker in example 6. For example,conjugates of a humanized antibody with DM1 were prepared using BMPS(N[β-maleimidopropyloxy]succinimide ester) or GMBS((N-[γ-maleimidobutyryloxy]succinimide ester) linker as shown in FIG.25. The initial reaction mixture containing BMPS or GMBS (8 mM) and DM1thiol (10.4 mM) in 60% DMA/40% (v/v) 200 mM succinate buffer, pH 5,showed complete reaction of maleimide moiety (based on decay ofmaleimide absorbance at 302-320 nm) when checked at 15 min. Thisreaction mixture was added, in two portions 30 min apart, to a humanizedantibody solution at 2.5 mg/ml in 80% aqueous EPPS buffer, pH 8.1,containing 20% DMA (v/v) with the total linker added at 8 molarequivalents to antibody. The conjugate mixture was gel purified after 4h and subjected to 2 rounds of dialysis. Conjugates with DM1/antibodyratio of 3.8 and 5.1 were prepared with 71-75% recovery, and highmonomer % (96.2-97.6%). These conjugates prepared with GMBS or BMPSshowed no unconjugated free drug by HISEP HPLC analysis. Similarconjugates containing non-cleavable linkers with linear alkyl chains canbe prepared using AMAS (N-[β-maleimidoacetoxy]succinimide ester) or EMCS(N-[β-maleimidocaproyloxy]succinimide ester) or thesulfo-N-hydroxysuccinimide esters (sulfo-GMBS, sulfo-EMCS) as shown inFIG. 25. Table 1 shows the monomer % for select conjugates prepared bythe method described in this invention, which all showed high monomer %by size-exclusion chromatography analysis. For comparison, monomer % arealso shown for conjugates prepared by the traditional two-stepconjugation method (by the initial reaction of antibody withheterobifunctional linker followed by reaction with mayansinoid thiol).

TABLE 1 Monomer % for select conjugates made by the method described inthis application versus by traditional two-step conjugation methodsConjugate D/A Conjugation method % Monomer Ab-PEG₄-Mal-DM1 6.6 thisinvention 99.0 Ab-PEG₄-Mal-DM1 6.8 two-step 98.0 Ab-Sulfo-Mal-DM1 3.6this invention 99.0 Ab-Sulfo-Mal-DM1 4.0 two-step 96.7 Ab-SMCC-DM1 4.0this invention 98.6 Ab-SMCC-DM1 3.8 two-step 97.0 Ab-PEG₄-Mal-DM4 6.2this invention 96.9 Ab-PEG₄-Mal-DM4 6.1 two-step 84.5 Ab-SPDB-DM4 4.1this invention 99.4 Ab-SPDB-DM4 3.9 two-step, one-pot 95.7

The invention claimed is:
 1. A process for preparing a purifiedconjugate in a solution, wherein the conjugate comprises a maytansinoidcomprising a thiol group linked to a cell binding agent, the processcomprising the steps of: (a) contacting a maytansinoid comprising athiol group with a bifunctional linker reagent to covalently attach thelinker to the maytansinoid and thereby prepare an unpurified firstmixture comprising the maytansinoid having linkers bound thereto, (b)conjugating a cell binding agent to the maytansinoid having linkersbound thereto by reacting the unpurified first mixture with the cellbinding agent to prepare a second mixture, and (c) subjecting the secondmixture to tangential flow filtration, dialysis, gel filtration,adsorptive chromatography, selective precipitation or a combinationthereof to thereby prepare the purified conjugate, wherein thebifunctional linker reagent is γ-maleimidobutyric acid N-succinimidylester (GMBS), and wherein step (b) is carried out in a solution at a pHfrom about 5 to about 8.7.
 2. The process of claim 1, wherein the pH ofthe solution in step (b) is from about 6.5 to about 8.5.
 3. The processof claim 1, wherein the second mixture in step (b) is substantially freeof undesired cross-linked, hydrolyzed species formed due tointramolecular or inter-molecular reactions.
 4. The process of claim 1,wherein the maytansinoid is DM1.
 5. The process of claim 1, wherein themaytansinoid is DM4.
 6. The process of claim 1, wherein the cell bindingagent is an interferon, interleukin 2 (IL-2), interleukin 3 (IL-3),interleukin 4 (IL-4), interleukin 6 (IL-6), insulin, EGF, TGF-a, FGF,G-CSF, VEGF, MCSF, GM-CSF, transferrin, or an antibody.
 7. The processof claim 6, wherein the cell binding agent is an antibody.
 8. Theprocess of claim 7, wherein the antibody is a monoclonal antibody. 9.The process of claim 7, wherein the antibody is a human or a humanizedmonoclonal antibody.
 10. The process of claim 7, wherein the antibody isMY9, anti-B4, C242, or an antibody that binds to an antigen selectedfrom EpCAM, CD2, CD3, CD4, CD5, CD6, CD11, CD19, CD20, CD22, CD26, CD30,CD33, CD37, CD38, CD40, CD44, CD56, CD79, CD105, CD138, EphA receptors,EphB receptors, EGFR, EGFRvIII, HER2, HER3, mesothelin, cripto,alphavbeta3, alphavbeta5, and alphavbeta6 integrin.
 11. The process ofclaim 9, wherein the human or the humanized antibody is huMy9-6, huB4,huC242, huN901, DS6, CNTO 95, B-B4, trastuzumab, pertuzumab,bivatuzumab, sibrotuzumab, rituximab, or a human or humanized antibodythat binds to an antigen selected from EphA2 receptor, CD38, and IGF-IR.12. The process of claim 7, wherein the antibody is a fragment of anantibody.
 13. The process of claim 12, wherein the fragment of anantibody is a scFv, Fab, Fab′ or F(ab′)2.
 14. The process of claim 1,wherein an excess of maytansinoid relative to the bifunctional linkerreagent is used.
 15. The process of claim 14, wherein the processfurther comprises the step of quenching the excess maytansinoid in theunpurified first mixture with a quenching reagent between steps (a) and(b).
 16. The process of claim 15, wherein the quenching reagent isselected from 4-maleimidobutyric acid, 3-maleimidopropionic acid,N-ethylmaleimide, iodoacetamide, and iodoacetamidopropionic acid.